EP2750689A2 - Dérivés d'apidaecine utilisés comme peptides antibiotiques - Google Patents

Dérivés d'apidaecine utilisés comme peptides antibiotiques

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Publication number
EP2750689A2
EP2750689A2 EP12774981.0A EP12774981A EP2750689A2 EP 2750689 A2 EP2750689 A2 EP 2750689A2 EP 12774981 A EP12774981 A EP 12774981A EP 2750689 A2 EP2750689 A2 EP 2750689A2
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EP
European Patent Office
Prior art keywords
amino acid
arginine
acid residues
cysteine
peptide
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EP12774981.0A
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German (de)
English (en)
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EP2750689B1 (fr
Inventor
Ralf Hoffmann
Daniel Knappe
Kai Hilpert
Ralf Mikut
Serge RUDEN
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Universitaet Leipzig
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AMP Therapeutics GmbH and Co KG
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43572—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from bees
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids

Definitions

  • This invention relates to modified antibiotic peptides, especially for use in medicine. Furthermore, the invention relates to compositions and methods for killing microorganisms, such as bacteria, viruses or fungi, and methods for treating microbial infections.
  • Inducible antibacterial peptides represent a field of research in which today's biochemistry, immunology and drug discovery meet.
  • Peptide antibiotics ranging in size from 13 up to more than one hundred amino acids, have been isolated from plants, animals and microbes (Boman, H.G. 1995).
  • a single animal has about 6-10 antimicrobial peptides, with each peptide often displaying a completely different spectrum of activity (Barra, D et al., 1998). It is known that the large number of antibacterial peptides, including the well-studied defensins, cecropins and magainins, act through a "lyric / ionic" mechanism. As a common mechanism of action of these "lyric" peptides, a permeabilizing effect on the bacterial cytoplasmic membrane is discussed. A cationic, amphipathic structure that forms hydrophilic ion (proton) channels in a lipid bilayer is the basis of this activity.
  • antimicrobial peptides that specifically recognize a bacterial protein or other intracellular or extracellular bacterial components without exhibiting cross-reactivity with mammalian analogs.
  • AMP antimicrobial peptides
  • proline-rich antimicrobial peptides including apidaecins, drosocin and pyrrhocoricin, originally isolated from insects.
  • proline-rich antimicrobial peptides including apidaecins, drosocin and pyrrhocoricin
  • proline-rich peptides eg, apidaecin, drosocin, and pyrrhocoricin
  • apidaecin e.g. apidaecin, drosocin, and pyrrhocoricin
  • DnaK heat shock protein DnaK
  • proline-rich peptides in sharp contrast to AMPs with defined secondary structure such as melittin or gramicidin, appear to be neither hemolytic nor toxic to eukaryotic cells in vitro.
  • Apidaecin is a peptide found in the honeybee hemolymph (Apis melliferd) and plays an important role in the defense against microbial infections. Studies have shown that apidaecin is mainly active against Gram-negative bacteria (Li, WF 2006). US 5,300,629 A discloses apidaecins of the general formula:
  • X is valine or isoleucine and Z is leucine or isoleucine.
  • WO 9523513 A discloses apidaecin derivatives with the sequence motifs where XI is isoleucine or leucine, X2 arginine or lysine, X3 threonine, glutamine or arginine, X4 tyrosine, glutamine or proline and X5 valine or alanine.
  • Czihal et al. In 2007, the apidaecin sequence was modified with natural and modified amino acids to increase antibacterial activity and protease resistance.
  • the abstract does not mention details of the modifications, in particular it does not mention sequences and gives no indication of which modifications are successful.
  • WO 2009/013262 A1 discloses peptides of the general formula: where Sub2 may contain Arg-Ile (RI).
  • Gobbo et al. 2006 discloses apidaecin peptoids in which the arginine residues (positions 4, 12 and 17) are replaced by corresponding N-substituted glycines. That is, the side chain in the apidaecin peptoids is shifted from the Ca to the Na atom. This reduces the protease sensitivity.
  • Table 1 compares general formulas and individual sequences of hitherto known apidaecin derivatives and related peptides in an alignment:
  • proline-rich antimicrobial peptides The action of proline-rich antimicrobial peptides is very complex because they must penetrate the cell membrane and penetrate into the cytoplasm to inhibit a particular intracellular bacterial target molecule, without, however, acting toxic to mammalian cells and blood cells. Another important issue is the stability of peptides or peptide derivatives (including peptidomimetics) to degradation by peptidases or proteases in blood and bacteria. Therefore, the ideal antibiotic peptide has high antibacterial activity (small MH values), no cell toxicity, no hemolytic activity and a half-life of several hours in blood.
  • the invention relates to a peptide for use as a drug against infection with Gram-positive bacteria or against infection by both Gram-positive and Gram-negative bacteria.
  • the present invention also relates to such a peptide or a pharmaceutical composition containing such a peptide, a peptide multimer comprising at least two such peptides, a nucleic acid coding for the peptide and a host cell containing the nucleic acid.
  • the peptide described here can also be used as an antibiotic against Gram-negative bacteria.
  • the peptide according to the invention is described below.
  • the object is achieved by the peptides according to FIG. 2, i. by those peptides of Figure 1 whose value is less than 1.
  • a value less than 1 means improved activity over native apidaecin.
  • the subject of this invention are the peptides shown in FIG.
  • the amino acid sequence of formula A or B has at least 60%, preferably at least 70%, preferably at least 80% amino acid sequence identity to native
  • Apidaecin lb according to SEQ ID NO: 2 on. According to the invention, in formula A or B:
  • X 1 is selected from nonpolar, aromatic, positively charged amino acid residues
  • N 2 , X 2 and P 5 are independently selected from neutral and positively charged amino acid residues
  • X 3 is selected from positively charged amino acid residues (preferably non-aromatic, especially not histidine), amino acid residues having a thiol group and amino acid residues having a selenol group;
  • V 6 is selected from non-polar amino acid residues having at least 2 C atoms, preferably 2 to 8 C atoms, in the side chain, aromatic amino acid residues, positively charged amino acid residues, thiol group amino acid residues, selenol group amino acid residues, proline and proline derivatives ;
  • Y 7 is selected from tyrosine, positively charged amino acid residues (preferably non-aromatic, especially not histidine), amino acid residues having a thiol group and amino acid residues having a selenol group;
  • I 8 is selected from nonpolar, aromatic amino acid residues having at least 2 and at most 8 C atoms in the side chain, positively charged amino acid residues, amino acid residues having a thiol group and amino acid residues having a selenol group;
  • P 9 , Pij , P 14 and P I 6 are independently selected from positively charged amino acid residues, thiol group amino acid residues, selenol group amino acid residues, nonpolar aromatic amino acid residues, heteroaromatic amino acid residues, proline and proline derivatives;
  • X 4 is selected from neutral, positively charged amino acid residues, amino acid residues having a thiol group and amino acid residues having a selenol group;
  • X 5 is selected from proline, proline derivatives, positively charged amino acid residues, amino acid residues having a thiol group, and amino acid residues having a selenol group;
  • R 12 is a positively charged amino acid residue (preferably non-aromatic, especially not histidine);
  • H 15 is selected from histidine, positively charged amino acid residues, amino acid residues having a thiol group, and amino acid residues having a selenol group;
  • X 6 is selected from positively charged amino acid residues, preferably X 6 (unchanged) is arginine;
  • X 7 is selected from non-polar amino acid residues (preferably with at least 2 C atoms in the side chain, preferably with 2-8 C atoms in the side chain), positively charged amino acid residues (preferably with at least 2 C atoms in the side chain, preferably with 2 - 8 C-atoms in the side chain), amino acid residues with a thiol group and amino acid residues with a selenol group.
  • N 2 is selected from non-polar amino acid residues, preferably nonpolar aromatic amino acid residues having from 6 to 15, preferably 8 to 15, C atoms in the side chain, positively charged amino acid residues, thiol group amino acid residues and selenol group amino acid residues, preferably N 2 selected from arginine, ornithine, lysine, cysteine, seleno-cysteine, valine, isoleucine, methionine, phenylalanine and tryptophan, more preferably arginine, ornithine, lysine, cysteine, seleno-cysteine, phenylalanine and tryptophan, at least one of the radicals selected from P 5 , V 6 , Y 7 , I 8 , P 9 , P ⁇ , P 14 and Pi 6 is a positively charged amino acid residue (preferably arginine, ornithine or lysine), an amino acid residue a thiol group or an
  • Hi 5 is selected from amino acid residues having a thiol group and amino acid residues having a selenol group (preferably cysteine or seleno-cysteine) and / or
  • X 7 is selected from positively charged amino acid residues, amino acid residues having a thiol group and amino acid residues having a selenol group, preferably X 7 is selected from cysteine, lysine, arginine and ornithine.
  • the peptide is characterized in that at least one of the positions 2, 5 to 11, 13 to 16 and 18 of SEQ ID no. 2 is modified so that the peptide according to formula A or B is at least one of the following conditions:
  • N 2 is selected from nonpolar amino acid residues, positively charged amino acid residues, amino acid residues having a thiol group and amino acid residues having a selenol group, preferably N 2 is selected from tryptophan, arginine, lysine and cysteine,
  • X 4 is selected from lysine, ⁇ -hydroxylysine, ⁇ - ⁇ -methyllysine, allo-hydroxylysine, cysteine and seleno-cysteine,
  • P 5 , V 6 , Y 7 , Is, P 9, Pn, P 11, P 14, Pia, and X 7 is a positively charged amino acid residue, an amino acid residue having a thiol group, or an amino acid residue containing a selenol Group, and / or
  • Hi 5 is selected from amino acid residues having a thiol group and amino acid residues having a selenol group, preferably H i5 is cysteine.
  • the peptide is characterized in that at least one of the following positions of SEQ ID no. 2 is modified so that the peptide according to formula A or B is at least one of the following conditions:
  • N 2 is selected from nonpolar amino acid residues, positively charged amino acid residues, amino acid residues having a thiol group and amino acid residues having a selenol group, preferably N 2 is selected from tryptophan, arginine, lysine and cysteine,
  • At least one of the radicals selected from P5, V 6, Y 7, 1 8, P 9, P] 3, Pu, P I6, and X 7 is a positively charged amino acid residue, an amino acid residue with a thiol group or an amino acid residue with a Selenol group, and / or
  • Hi 5 is selected from amino acid residues having a thiol group and amino acid residues having a selenol group, preferably H
  • the peptide is characterized in that at least one of the following positions of SEQ ID no. 2 is modified so that the peptide according to formula A or B is at least one of the following conditions:
  • N 2 is arginine or glutamine
  • P5 is cysteine or arginine
  • At least one of the radicals selected from I 8 , P 13 , P 14 , X 7 is arginine, and / or
  • X 4 is arginine.
  • X 7 is preferably selected from positively charged amino acid residues, amino acid residues having a thiol group and amino acid residues having a selenol group, more preferably X 7 is selected from cysteine, lysine, arginine and ornithine.
  • 1, 2, 3, 4 or all 5 conditions are met.
  • the conditions of N 2 and X 4 may be satisfied; or that of N 2 and at least one of P 5 , V 6 , Y 7 , I 8 , P 9 , P 11 , P 13 , P 14 , P 16 , and X 7 ; or those of N 2 and H 15 ; or those of N 2 , X 4 and at least one of P 5 , V 6 , Y 7 , I 8 , P 9 , P 11 , P 13 , P I4 , P 16 , and X 7 .
  • the erfmdungshiele peptide thus contains no negatively charged amino acid residues.
  • a peptide according to the invention which contains an amino acid sequence according to formula A or B and in which N 2 and / or at least one of P 5 , V 6 , Y 7 , I 8 , P 9 , P 11 , P 13 , P 14 and / or P 16 is as selected above and wherein: H 15 is selected from positively charged, non-aromatic amino acid residues, amino acid residues having a thiol group and amino acid residues having a selenol group, preferably cysteine, lysine, arginine or ornithine , Further preferred is a peptide according to the invention which contains an amino acid sequence according to formula A or B and in which at least one of P 5 , V 6 , Y 7 , I 8 , P 9 , P 11 , P 13 , P 14 and / or P 16 is selected as above and wherein: N 2 is selected from positively charged, non-aromatic amino acid residues, amino acid residues having a thiol group and
  • the invention is based on a substitution analysis of Apidaecin Ib:
  • the substitution analysis showed that the positions Asn2, Asn3, Pro5 in the native peptide are particularly unfavorably occupied and the antibiotic activity can be optimized by substitution with any other non-negatively charged amino acids.
  • the antibiotic activity can be improved in particular by the substitution with positively charged amino acid residues (such as lysine or arginine) or by amino acid residues with a thiol group (such as cysteine).
  • positively charged amino acid residues such as lysine or arginine
  • amino acid residues with a thiol group such as cysteine
  • peptides with improved antimicrobial activity identified in the substitution analysis were further tested for their antibacterial activity against different bacterial strains, especially Gram-positive bacteria. It was found that the peptides according to the invention are also advantageously antibacterial active against Gram-positive bacteria, such as S. aureus.
  • Preferred peptides of the invention contain at least one additional amino acid residue X 6 and optionally another amino acid residue X 7 at its C-terminus, wherein X 6 is selected from positively charged amino acid residues and wherein X 7 is selected from positively charged amino acid residues, amino acid residues having a thiol group and Amino acid residues with a selenol group.
  • Such a preferred peptide comprises at least one amino acid sequence according to the general formula B or C:
  • peptides of the formula C according to the invention additionally contain the further amino acid residue X 6 , which is as defined above.
  • X 6 is arginine.
  • Preferred peptides according to the invention are apidaecin derivatives which have at least one of the following mutations in the amino acid sequence of apidaecin 1b (SEQ ID NO: 2), wherein the numbers relate to the positions within the amino acid sequence according to SEQ ID NO: 2:
  • the peptides according to the invention preferably have at least 16 amino acid residues, preferably at least 18 and preferably up to 50 amino acid residues.
  • the peptide according to the invention contains no negatively charged amino acid residues.
  • a negatively charged amino acid residue according to the invention contains a negatively charged amino acid side chain under physiological conditions.
  • physiological conditions mean a pH of 7.4, a temperature of 37 ° C. and an osmotic pressure of 300 mosmol / kg.
  • a positively charged amino acid residue according to the invention contains a positively charged amino acid side chain under physiological conditions.
  • Positively charged radicals are preferably non-aromatic and are preferably selected from arginine, lysine, ⁇ -hydroxylysine, homoarginine, 2,4-diaminobutyric acid, ⁇ -homoarginine, D-arginine, arginal (COOH in arginine is replaced by -CHO), 2- Amino-3-guanidinopropionic acid, 2-amino-4-guanidinobutyric acid, nitroarginine (preferably N (G) -nitroarginine), nitrosoarginine (preferably N (G) -nitrosoarginine), methylarginine (preferably N-methyl-arginine), ⁇ - ⁇ - Methyllysine, alloxylysine, 2,3-diaminopropionic acid, 2,2'-diaminopimelic acid, ornithine, sym-dimethylarginine, asym-dimethylarginine, 2,6-diamino
  • a neutral amino acid residue contains an uncharged amino acid side chain under physiological conditions. Neutral amino acid residues are thus neither positively nor negatively charged under physiological conditions.
  • the term neutral amino acid residues includes polar and nonpolar amino acid residues.
  • a polar amino acid residue has at least one polar group in the amino acid side chain. These polar groups are uncharged under physiological conditions and selected from hydroxyl, sulfhydryl, amine, amide, and ester groups as well as other groups that allow the formation of hydrogen bonds.
  • Preferred neutral polar amino acid residues are selected from asparagine, cysteine, glutamine, serine, threonine, tyrosine, citrulline, N-methylserine, homoserine, allo-threonine, 3,5-dinitrotyrosine and ⁇ -homoserine.
  • a nonpolar (or even hydrophobic) amino acid residue has no polar groups and contains an uncharged amino acid side chain under physiological conditions, preferably with a hydropathic index above 0, more preferably above 3.
  • Preferred nonpolar hydrophobic side chains are selected from H, alkyl, alkylene - Alkoxy, alkenoxy, alkylsulfanyl and Alkenylsulfanylresten having 1 to 10, preferably 2 to 6 C-atoms, and aryl radicals having 5 to 12 carbon atoms.
  • Preferred amino acid residues having a non-polar, hydrophobic side chain are selected from glycine, alanine, leucine, isoleucine, valine, methionine, alanine, phenylalanine, tryptophan, N-methylleucine, tert-butylglycine, cyclohexylalanine, ⁇ -alanine, 1-aminocyclohexylcarboxylic acid, N- Methylisoleucine, norleucine, norvaline and N-methylvaline.
  • Aromatic amino acid residues have at least one aryl or heteroaryl ring.
  • the term includes polar and nonpolar aromatic amino acid residues, where polar and nonpolar are as defined above.
  • Preferred polar aromatic amino acid residues are selected from aryl radicals having 5 to 12 C atoms, these radicals bearing at least one polar group, as well as heteroaromatic amino acid residues.
  • Preferred heteroaromatic amino acid residues are heteroaryl residues having 3 to 10 C atoms and 1 to 4 heteroatoms (preferably ⁇ , S or O) in the ring system, particularly preferably histidine.
  • Particularly preferred polar aromatic amino acid residues are selected from tyrosine, 3,5-dinitrotyrosine, histidine and histidine derivatives.
  • histidine derivative stands for a histidine-derived amino acid residue which is preferably obtained from histidine by structural modification of preferably one or two functional groups.
  • Preferred histidine derivatives are C 1 -C 3 -alkylated (preferably ⁇ -alkyl) histidines, in particular ⁇ -methyl-histidine.
  • Preferred nonpolar aromatic amino acid residues are selected from aryl radicals having 5 to 12 C atoms, these radicals bearing no polar groups.
  • nonpolar aromatic amino acid residues are tryptophan, phenylalanine, phenylglycine, homophenylalanine, 4-tert-butylphenylalanine, methyltryptophan, naphthylalanine, diphenylalanine, methylphenylalanine, phenyl-phenylalanine and benzoylphenylalanine.
  • Amino acid residues having a thiol group or selenol group are preferably selected from alkyl-alkoxy, alkenoxy, alkylsulfanyl and alkenylsulfanyl radicals having 1 to 10, preferably 2 to 6, C atoms, or aryl radicals having 5 to 12 C-atoms, which at least one free (unsubstituted) thiol group (-SH) or selenol (-SeH) group carry.
  • Particularly preferred amino acid residues having a thiol group or selenol group are cysteine and seleno-cysteine.
  • proline derivative stands for an amino acid residue derived from proline, which is preferably obtained from proline by structural modification of preferably exactly one or two functional groups.
  • Preferred proline derivatives are selected from ⁇ -cyclohexylalanine, 3,4-cis-methanoproline, 3,4-dehydroproline, hydroxyproline, mercaptoproline, thioproline, fluoroproline and homoproline.
  • hydroxyproline includes, inter alia, cis-4-hydroxyproline, trans-4-hydroxyproline, cis-3-hydroxyproline and trans-3-hydroxyproline.
  • hydroxyproline derivative is correspondingly an hydroxyproline-derived amino acid residue obtained from hydroxyproline, preferably by structural alteration of a functional group.
  • Preferred hydroxyproline derivatives are selected from hydroxy- ⁇ -cyclohexylalanine and the above-mentioned proline derivatives which are substituted with a hydroxyl group.
  • the amino acid residue X 1 is preferably selected from arginine, lysine, ⁇ -hydroxylysine, homoarginine, 2,4-diaminobutyric acid, ⁇ -homoarginine, D-arginine, arginal, 2-amino 3-guanidinopropionic acid, nitroarginine, N-methyl-arginine, ⁇ - ⁇ -methyllysine, allo-hydroxy lysine, 2,3-diaminopropionic acid, 2,2'-diaminopimelic acid, ornithine, sym-dimethylarginine, asym-dimethylarginine, 2,6- Diaminohexanoic acid, p-aminobenzoic acid, 3-aminotyrosine, glycine, alanine, valine, isoleucine, leucine, methionine, N-methylleucine,
  • X selected from nonpolar amino acid residues is preferably alanine, glycine, phenylalanine, methionine, isoleucine, valine, leucine and proline, especially leucine and proline, and aromatic amino acid residues such as tryptophan and tyrosine and less preferably histidine, and most preferably positively charged residues , especially lysine, arginine, and cysteine.
  • X selected from cysteine, lysine, arginine and ornithine or else unchanged glycine.
  • amino acid residues N 2 , X 2 and P 5 are selected from all non-negatively charged amino acids. Glycine and alanine for X2 for P 5 less preferred.
  • the amino acid residues N 2 , X 2 and P 5 are preferably independently selected from arginine, lysine, ⁇ -hydroxylysine, homoarginine, ⁇ -homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid, 2-amino-3-guanidinopropionic acid, Nitroarginine, nitrosoarginine, N-methyl-arginine, ⁇ - ⁇ -methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2'-diaminopimelic acid, ornithine, sym-dimethylarginine, asym-dimethylarginine, 2,6-diaminohexynic acid , p-aminobenzoic acid, 3-aminotyrosine, asparagine, cysteine, seleno-cysteine, glutamine, serine, threonine, citrulline, N
  • N 2 is particularly preferably selected from cysteine, tryptophan, phenylalanine, lysine, arginine and ornithine or also unchanged asparagine.
  • X 2 is particularly preferably selected from cysteine, lysine, arginine, ornithine and homoarginine or also unchanged asparagine.
  • P 5 is particularly preferably selected from cysteine, lysine, arginine, ornithine, histidine and tryptophan or also unchanged proline.
  • the amino acid residue X 3 is selected from arginine, lysine, ⁇ -hydroxylysine, homoarginine, ⁇ -homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid, ⁇ -homoarginine, 2 Amino-3-guanidinopropionic acid, nitroarginine, nitrosoarginine, N-methyl-arginine, ⁇ - ⁇ -methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2'-diaminopimelic acid, ornithine, sym-dimethylarginine, asym-dimethylarginine, 2 , 6-diaminohexanoic acid, p-aminobenzoic acid, cysteine and seleno-cysteine.
  • X 3 is particularly preferably selected from
  • the amino acid residue V 6 is selected from arginine, lysine, ⁇ -hydroxylysine, homoarginine, ⁇ -homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid, ⁇ -homoarginine, 2- Amino-3-guanidinopropionic acid, nitroarginine, nitrosoarginine, N-methyl-arginine, ⁇ - ⁇ -methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2'-diaminopimelic acid, ornithine, sym-dimethylarginine, asym-dimethylarginine, 2 , 6-diaminohexanoic acid, p-aminobenzoic acid, valine, isoleucine, leucine, methionine, N-methylleucine,
  • V 6 is selected from positively charged amino acid residues, preferably arginine and lysine, and cysteine, and less preferably from aromatic residues such as histidine, N-methyl histidine, tryptophan, 3,5-dinitrotyrosine and tyrosine, as well as nonpolar residues (with more than two carbon atoms), such as isoleucine and proline.
  • V 6 is particularly preferably selected from arginine, lysine, cysteine and tryptophan or else unchanged valine.
  • the amino acid residue Y 7 is selected from tyrosine, arginine, lysine, ⁇ -hydroxylysine, homoarginine, ⁇ -homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid, ⁇ -homoarginine, 2-amino-3-guanidinopropionic acid, nitroarginine, nitrosoarginine, N-methyl-arginine, ⁇ - ⁇ -methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2'-diaminopimelic acid, ornithine, sym-dimethylarginine, asym-dimethyl - arginine, 2,6-diaminohexynoic acid, p-aminobenzoic acid, cysteine and seleno-cysteine.
  • Y7 is selected from positively charged amino acid residues, such as arginine and lysine, and cysteine.
  • Y 7 is particularly preferably selected from arginine, ornithine, lysine and cysteine or also unchanged tyrosine.
  • the amino acid residue I 8 is selected from arginine, lysine, ⁇ -hydroxylysine, homoarginine, ⁇ -homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid, 2-amino-3 guanidinopropionic acid, nitroarginine, nitrosoarginine, N-methyl-arginine, ⁇ - ⁇ -methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2'-diamino-pimelic acid, ornithine, sym-dimethylarginine, asym-dimethylarginine, 2,6 Diaminohexaenoic acid, p-aminobenzoic acid, 3-aminotyrosine, cysteine, seleno-cysteine, valine, isoleucine, leucine, N-methyl-
  • I 8 is selected from positively charged amino acid residues such as arginine and lysine, and cysteine and somewhat less preferably aromatic residues (having a maximum of 8 C atoms) such as histidine, phenylalanine and tyrosine.
  • I 8 is particularly preferably selected from arginine, ornithine, lysine, histidine and cysteine or also unchanged isoleucine.
  • peptides of the formulas A, B or C are the amino acid residues P 9 , Pi3 , P
  • amino acid residues P 9 , P, 3 , P ! 4 and P I 6 are independently selected from positively charged amino acid residues such as arginine and lysine, and cysteine, and in the case of P 9 also less preferably heteroaromatic residues such as histidine and nonpolar aromatic amino acid residues, phenylalanine and tryptophan.
  • the amino acid residues P 9 , P 13, P [ and P J6 are particularly preferably selected independently of one another from arginine, ornithine, lysine and cysteine or else unchanged proline.
  • the amino acid radical X 4 is selected from arginine, lysine, ⁇ -hydroxylysine, homoarginine, ⁇ -homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid, 2-amino-3 guanidinopropionic acid, nitroarginine, nitrosoarginine, N-methyl-arginine, ⁇ - ⁇ -methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2'-diamino-pimelic acid, ornithine, sym-dimethylarginine, asym-dimethylarginine, 2,6 Diaminohexaenoic acid, p-aminobenzoic acid, 3-aminotyrosine, cysteine, seleno-cysteine, glutamine, citrulline, isoleucine,
  • X 4 is selected from positively charged amino acid residues such as arginine and lysine, and cysteine, and somewhat less preferably isoleucine, leucine, histidine, and phenylalanine.
  • X 4 is particularly preferably selected from arginine, ornithine, lysine, histidine and cysteine or, unchanged, glutamine.
  • the amino acid residue X 5 is selected from arginine, lysine, ⁇ -hydroxylysine, homoarginine, ⁇ -homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid, 2-amino-3 guanidinopropionic acid, nitroarginine, nitrosoarginine, N-methyl-arginine, ⁇ - ⁇ -methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2'-diamino- Pimelic acid, ornithine, sym-dimethylarginine, asym-dimethylarginine, 2,6-diaminohexanoic acid, p-aminobenzoic acid, 3-aminotyrosine, cysteine, proline, ⁇ -cyclohexylalanine, 3,4-cis-
  • X 5 is selected from positively charged amino acid residues such as arginine and lysine, and cysteine, and somewhat less preferably nonpolar aromatic amino acid residues such as phenylalanine and tryptophan.
  • X 5 is particularly preferably selected from arginine, ornithine, lysine, histidine and cysteine or also unchanged glutamine.
  • the amino acid residue R ] 2 is selected from arginine, lysine, ⁇ -hydroxylysine, homoarginine, ⁇ -homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid, 2-amino-3 -guanidinopropionic acid, nitroarginine, nitrosoarginine, N-methyl-arginine, ⁇ - ⁇ -methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2'-diamino-pimelic acid, ornithine, sym-dimethylarginine, asym-dimethylarginine, 2, 6-diaminohexanoic acid, p-aminobenzoic acid and 3-aminotyrosine.
  • R n is particularly preferably arginine, homoarginine, ornithine or
  • the amino acid residue H 15 is selected from histidine, N-methyl-histidine, arginine, lysine, ⁇ -hydroxylysine, homoarginine, ⁇ -homoarginine, D-arginine, arginal, 2,4- Diaminobutyric acid, 2-amino-3-guanidinopropionic acid, nitroarginine, nitrosoarginine, N-methyl-arginine, ⁇ - ⁇ -methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2'-diaminopimelic acid, ornithine, sym-dimethylarginine, asym Dimethylarginine, 2,6-diaminohexanoic acid, p-aminobenzoic acid and 3-aminotyrosine and cysteine.
  • arginine, homoarginine, ornithine, lysine, cysteine or, unchanged, histidine is particularly preferred.
  • 1 to 10, more preferably at most 7, most preferably a maximum of 5 amino acid residues are changed from the native amino acid sequence of apidaecin 1b (SEQ ID NO: 2) as described above.
  • the remaining amino acid residues correspond to the respective amino acid at the corresponding position in the native Apidaecin Ib (SEQ ID NO: 2).
  • the peptide of the invention contains at least 3, especially at least 4 positively charged amino acid residues, more preferably 5 to 10 positively charged amino acid residues.
  • the peptide contains at least 4, more preferably at least 5, and preferably at most 8 proline residues. More preferably, the peptide contains at least one cysteine residue, preferably at most three cysteine residues.
  • Preferred peptides according to the invention contain at least one amino acid sequence according to one of the general formulas 1 to 10, where the radicals Xi to X 4 and N 2 are those described above for the formulas A, B and C have the meanings mentioned and the remaining amino acid residues correspond to the IUPAC one-letter code:
  • CT is the free C-terminal carboxyl group of the C-terminal amino acid of the peptide (-COOH), its modified C-terminal carboxyl group or a peptide having preferably 2 to 6 amino acid residues with a free or modified C-terminus.
  • CT is a dipeptide, more preferably having one of the following amino acid sequences RF (Arg-Phe), RL (Arg-Leu), RC (Arg-Cys), RK (Arg-Lys) or RR (Arg-Arg), with a free or modified C-terminus.
  • a peptide according to the invention of the general formulas A ', B' or C at least the N-terminus of the amino acid residue X 1 or the C-terminus of the peptide is modified.
  • a "modification" of the N-terminal amino group or of the C-terminal carboxyl group is understood to mean that the amino group or the carboxyl group are modified, such as, for example, reduced or substituted.
  • NT thus represents the free N-terminus of amino acid X 1 or a modification of the N-terminal amino group (which replaces the N-terminal amino group of amino acid X 1 with NT) having the general formula NR 1 R 2.
  • reporter groups preferably fluorescent dyes (preferably fluorescein, Alexa488) and biotin;
  • a linker for linking to the modification of the C-terminus of the peptide of the general formula COR 3 (definition see below), to form a cyclic peptide, preferably based on guanidine, ethylene glycol oligomers, 2,4-diaminobutyric acid, 2,3- Diaminopropionic acid, 2,2'-diaminopimelic acid, desmosine or isodesmosine;
  • (V) linkers for the coupling of another peptide (Y 2 ) via a specific chemical or enzymatic reaction, preferably based on iodo, bromo or chloroalkanoic acids (eg iodoacetic acid) or maleimide for coupling to a thiol-containing peptide or another reactive Group (eg amino group, thiol group) for the coupling of a second peptide or peptide derivative (eg as active ester, aldehyde or thioester) as a carrier or carrier protein,
  • a specific chemical or enzymatic reaction preferably based on iodo, bromo or chloroalkanoic acids (eg iodoacetic acid) or maleimide for coupling to a thiol-containing peptide or another reactive Group (eg amino group, thiol group) for the coupling of a second peptide or peptide derivative (eg as active ester, aldehyde
  • N-terminal modifications in a peptide according to the invention are acetylation, formylation and guanidation of the N-terminus.
  • the N-terminus of a peptide according to the invention is guanidated.
  • the N-terminus is tetramethyl guanidated.
  • CT represents the free C-terminal carboxyl group of the C-terminal amino acid (-COOH) or a modified C-terminal carboxyl group of a peptide according to the invention.
  • the modified C-terminal carboxyl group CT preferably has the general formula COR 3 (R 3 replaces the hydroxyl group of the last amino acid) or Y1-COR 3 .
  • Y is an additional, preferably neutral polar or positively charged, amino acid residue, particularly preferably leucine, arginine or glutamine or a peptide, preferably having two to six amino acids, in particular two to four amino acid residues, preferably a dipeptide having the sequence ⁇ 6 ⁇ ?
  • Y * are selected from RI (Arg-Ile), RL (Arg-Leu), RV (Arg-Val), RC (Arg-Cys) and RR (Arg-Arg).
  • a preferred C-terminal modification of a peptide according to the invention is preferably selected from:
  • R 3 is a free hydroxyl group
  • esters R 3 is an alkoxy group, preferably methoxy, ethoxy, propoxy, iso-propoxy or butoxy
  • amides R 3 is an amine, preferably alkylamine, dialkylamine, methylamine, Ethylamine, dimethylamine or cyclohexylamine
  • imides R 3 is an amine to which another acid group, in particular the C-terminus of another peptide, for example as mentioned for Y 2 , is bonded to an acid group of a polymer or a carrier );
  • R 3 is an additional branched amino acid to form a dimer or oligomer structure, in particular selected from lysine, hydroxylysine, ornithine, 2,4-diaminobutyric acid, 2,3- Diaminopropionic acid, 2,2'-diaminopimelic acid, desmosine, isodesmosine and peptides (preferably with 2-4 amino acids) containing a combination of the aforementioned amino acids,
  • Linker for the coupling of another peptide (Y 2 ) via a specific chemical or enzymatic reaction, preferably based on iodo, bromo or chloroalkanoic acids (eg iodoacetic acid) or maleimide for coupling to a thiol-containing peptide or another reactive Group (eg amino group, thiol group) for the coupling of a second peptide or peptide derivative (eg as active ester, aldehyde or thioester) as a carrier or carrier protein,
  • a specific chemical or enzymatic reaction preferably based on iodo, bromo or chloroalkanoic acids (eg iodoacetic acid) or maleimide for coupling to a thiol-containing peptide or another reactive Group (eg amino group, thiol group) for the coupling of a second peptide or peptide derivative (eg as active ester, aldehyde or thi
  • Other peptide derivatives can be formed by modifications of the N-terminal or C-terminal ends of the peptides.
  • a modification of the C-terminus by means of thioester synthesis and subsequent substitution with primary amines.
  • the peptides of the invention are derived from native apidaecin 1b (according to SEQ ID NO: 2) by replacing (substituting) at least one and preferably at most 10 amino acids of the native sequence with another amino acid.
  • a peptide according to the invention preferably has an amino acid sequence in which there is a substitution with respect to SEQ ID No. 2 at at least one of the positions 2, 5 to 9 and 13 to 16 of SEQ ID No. 2.
  • a peptide according to the invention has an amino acid sequence in which there is a substitution with respect to SEQ ID No. 2 at at least one of the positions 2, 5 and 13 to 16 of SEQ ID No. 2, very particularly preferably at the positions 2 and 13
  • the peptide according to the invention particularly preferably comprises an amino acid sequence in which, in comparison to SEQ ID NO
  • a nonpolar amino acid residue preferably an aromatic amino acid residue having from 6 to 15, preferably 8 to 15, C atoms in the side chain, a positively charged amino acid residue, an amino acid residue having a thiol group or an amino acid residue having a selenol group; preferably selected from arginine, lysine, cysteine and tryptophan, very particularly preferably tryptophan, and / or
  • positions 5 to 9 or 13, 14 and 16 preferably at least one of positions 5, 13, 14 and 16, more preferably at position 13, a positively charged amino acid residue, an amino acid residue having a thiol group or an amino acid residue with a selenol group, preferably a positively charged amino acid residue, more preferably arginine and / or
  • a peptide according to the invention has an amino acid sequence in which, compared to SEQ ID No. 2 at position 2, a substitution with cysteine or tryptophan and / or at least one of positions 5 and 13 to 16 of SEQ ID No. 2 at least one substitution Cysteine, lysine or arginine is located.
  • a peptide according to the invention preferably has an amino acid sequence in which ornithine is additionally opposite SEQ ID No. 2 at position 1.
  • peptides according to the invention in particular those with the abovementioned substitutions at positions 2, 13, 14 and / or 18 of SEQ ID NO: 2, (in particular N2W, P13R, P14C and L18R) show a marked increase in activity over Pseudomonas aeruginosa and even against the Gram positive bacterium Staphylococcus aureus (compared to Apidaecin lb).
  • the antimicrobial effect also occurs at physiological salt concentrations.
  • the peptide has an amino acid sequence in which at least one of the following substitution is opposite to SEQ ID NO: 2:
  • P 5 Aliphatic amino acid without carboxyl group except alanine; aromatic amino acid, and / or
  • V 6 aromatic amino acid; heterocyclic amino acid, amino acid with positive residue;
  • Particularly preferred peptides according to the invention contain one of the amino acid sequences according to SEQ ID no. 9 to 43 from Table 2, one of the amino acid sequences according to SEQ ID no. 9 to 43 with a modified, preferably guanidated or acylated, N-terminus and / or with a modified, preferably amidated, C-terminus.
  • Particularly preferred peptides according to the invention contain one of the amino acid sequences from Table 2, wherein additionally the C-terminus of the peptide is amidated and / or the N-terminus is guanidated or tetramethyl-guanidated (referred to as "gu" in Tables 3 and 6). Preferred examples of such amidated peptides are listed in Table 3:
  • Preferred peptides with a guanidated N-terminus according to the invention comprise an amino acid sequence according to one of SEQ ID NO. 44 to SEQ ID NO. 49, wherein preferably additionally the C-terminus of the peptide is amidated.
  • All natural amino acids, unnatural amino acids or amino acid derivatives (such as imino acids) which form the peptides or peptide derivatives of the present invention may exist in either the L or D conformation. Unless otherwise specified, however, the building blocks in the sequences are preferably in the L-conformation.
  • the modifications of the N and C termini allow the coupling of the peptides to other groups, such as other amino acid sequences (possibly creating multimeric peptides or proteins) or other biomolecules that function as a carrier or label, for example Y 2 over NT.
  • the molecule acts as a carrier to combat bacterial infection in mammalian cells or to transport the antibacterial peptide and peptide derivative into bacteria which the antibacterial peptide can not enter alone (eg Gram-positive bacteria).
  • cell penetrating peptides are, for example, penetrains, Tat peptides, amphipathic model peptides (model amphipathic peptides) and transportanes.
  • target molecules are, for example, molecules that are known to bind to lipopolysaccharide (LPS) molecules that form the outside of Gram-negative bacteria.
  • LPS lipopolysaccharide
  • Known compounds for this application are, for example, anchor peptides, such as the AcmA motif from Lactobacillus or an antibody directed against lipopolysaccharide. The latter variant is preferred because it also has an intrinsic antibiotic effect and therefore can be used to increase the activity of the peptides of the invention.
  • the activity against Gram-negative and Gram-positive bacteria can be increased or extended the spectrum of action on other Gram-positive and Gram-negative bacteria and on the other hand introduced the antimicrobial peptides in mammalian cells so that even in these cells hidden bacteria, fungi or viruses can be achieved.
  • Part of the invention is the coupling of penetratin via a thioether bridge. The C terminus of penetraine was extended by one cysteine and coupled to the N-terminally labeled with iodoacetic acid antimicrobial peptide.
  • peptide as used herein means a sequence of amino acids linked by a peptide bond, wherein the amino acids are preferably selected from the twenty proteinogenic amino acids and wherein the amino acids in the L-configuration or D-configuration, or, in the case of isoleucine and threonine, also in the D-allo configuration (inversion of only one of the two chiral centers), including peptide derivatives which have been altered by substitution and / or modification of one or more amino acid residues by chemical groups, these chemical groups are other than the natural protein-forming amino acid residues, such as non-proteinogenic ⁇ -amino acids, ⁇ -amino acids or altered backbone peptides
  • altered backbone means that at least one peptide bond is chemically modified, i. H. is replaced by a non-cleavable bond under physiological conditions which can not be cut by endoproteases.
  • the non-cleavable bond is a modified peptide bond such as.
  • An alkylated amide bond is one either the nitrogen (N-alpha) or carbon atom (C-alpha) alkylated peptide bond.
  • the alkyl radical preferably has 1 to 3 C atoms. An example is N-methylation.
  • altered backbone includes other groups that are capable of forming a covalent bond with both the COOH group of the preceding amino acid residue and the NH 2 group of the following amino acid residue, and therefore do not necessarily maintain the peptide backbone structure.
  • groups that are capable of forming a covalent bond with both the COOH group of the preceding amino acid residue and the NH 2 group of the following amino acid residue, and therefore do not necessarily maintain the peptide backbone structure.
  • Sugar amino acid dipeptide isosters such as , Sugar amino acid dipeptide isosters, azapeptides, 6-homopolymers, gamma-peptides, depsipeptides (ester backbone bridges), Y-lactam analogs, ohgo (phenylene-ethylene) s, vinylogous sulfone peptides, poly-N-substituted glycines or oligocarbamates.
  • the peptide bond is preferably replaced by a non-cleavable for proteases bond.
  • This non-cleavable bond is preferably selected from the group of reduced amide bonds, alkylated amide bonds or thioamide bonds.
  • the peptides according to the invention are preferably linear.
  • the peptides of the invention are also cyclic, preferably the first (N-terminus) and the last amino acid (C-terminus) are linked via a peptide bond or a linker.
  • peptides or their peptide derivatives according to the invention can be produced either synthetically or, where applicable, recombinantly by conventional methods.
  • the peptides or peptide derivatives of this invention are conventionally prepared by the known synthetic techniques as described, for example, by Merrifield.
  • the peptides described in this invention are prepared by recombinant techniques by cloning a DNA fragment containing a nucleic acid sequence encoding one of the above-described peptides, and e.g. B. expressed in a microorganism or a host cell.
  • the coding nucleic acid sequences can be prepared synthetically or obtained by site-directed mutagenesis of an existing nucleic acid sequence.
  • the coding sequence thus prepared can be amplified from the RNA (or DNA) with appropriately prepared primers in a polymerase chain reaction (PCR) by known techniques. After purification, for example by means of agarose gel electrophoresis, the PCR product is ligated into a vector and finally transformed the host cell with the appropriate recombinant plasmid. Recombinant techniques are known for different host cells, for example E.
  • the peptides of this invention can be isolated from the host cells, either with classical cell disruption techniques or from the cell medium by conventional methods, eg, liquid chromatography, especially affinity chromatography.
  • the peptide according to the invention can be expressed as a single peptide or as an oligomer.
  • the oligomers may contain a plurality of peptide sequences which are linked via the N- or C-termini, or even contain an N- or C-terminal tag, which allows easier purification of the recombinant peptides or protein constructs.
  • Conventional molecular biology techniques and site-specific mutagenesis can be used to further alter the sequence to obtain the desired non-native peptide sequences. These recombinant techniques have already been applied to many antimicrobial peptides including apidaecin (see eg Maeno M et al., 1993).
  • the peptides can be isolated from the host cell culture or the in vitro translation system. This can be accomplished with the standard protein purification and isolation techniques known in the art. Such techniques may include, for example, immunoadsorption or affinity chromatography. It is also possible to tag the peptides during synthesis (e.g., histidine tag), which allows for rapid binding and purification. The tag can be subsequently enzymatically cleaved to obtain the active peptide sequence.
  • histidine tag e.g., histidine tag
  • the method can first be applied to the similar peptide to subsequently chemically or enzymatically convert it into the desired peptide or peptidomimetics in one or more steps to convict.
  • the invention also encompasses nucleic acids which code for the peptides according to the invention and, preferably non-human, host cells which contain a nucleic acid according to the invention.
  • the host cells are preferably selected as described above and do not include human embryonic stem cells.
  • the peptides according to the invention can be used individually, in combination, as multimers or as branched multimers. Useful combinations of the peptides of the invention include dendrimers and concatamers in which the peptides according to the invention are serially linked to one another or via spacers, eg. In the form of a peptide dimer or a peptide trimer, etc., by lining up the individual peptides.
  • This multimer can be composed of peptides or peptide derivatives having identical sequences or different sequences according to formula A or B.
  • the modified peptides can additionally be coupled to a biocompatible protein, for example human serum albumin, humanized antibodies, liposomes, micelles, synthetic polymers, nanoparticles and phages.
  • a biocompatible protein for example human serum albumin, humanized antibodies, liposomes, micelles, synthetic polymers, nanoparticles and phages.
  • multimers in which the peptides or peptide derivatives of the invention are individually combined can be prepared in the form of dendrimers or clusters, with three or more peptides attached to a center.
  • multiple peptides may be prepared as a multimeric construct or array.
  • optional amino acids eg, Gly-Ser-
  • spacers linker peptides
  • amino acids or other chemical compounds can be attached to the N- or C-terminus to link two or more peptides together or to couple to a carrier .
  • This arrangement may take the form of one or more of the synthetic peptides described above coupled to a carrier protein.
  • an array contains multiple peptides, each expressed as a multiple antigenic peptide, optionally coupled to a carrier protein.
  • the selected peptides are sequentially linked and expressed as a recombinant protein or polypeptide.
  • multiple peptides are sequentially linked, with or without amino acids as spacers (linker peptide) therebetween, to obtain a larger recombinant protein.
  • the recombinant protein can be fused to a carrier protein.
  • the multimeric constructs contain at least two peptides, one peptide being coupled to the other peptides via any amino acid. Any number of additional peptides may be added to any other amino acids of these peptides.
  • the second or the further peptides are coupled to a branched skeleton of the other peptides of the basic structure.
  • each additional peptide is covalently linked via the NT or CT group to another peptide of the assembly.
  • a multimeric construct or assembly having at least two peptides at least one or more peptides are attached to a support.
  • one or more of said peptides is a synthetic peptide fused to a carrier protein.
  • the peptides or polypeptide are either coupled to the same carrier or different peptides can be individually coupled as peptides to one or different immunologically inert carrier proteins.
  • Suitable carriers may enhance stability, presentation, or production, or alter the spectrum of activity of the peptides.
  • examples of carriers are human albumin, polyethylene glycol or other biopolymers or other naturally or non-naturally occurring polymers.
  • the major component is preferably a protein or other molecule that can increase peptide stability. An experienced person can simply select a suitable coupling unit.
  • the peptides are arranged in the form of a multiple antigenic peptide (MAP).
  • MAP multiple antigenic peptide
  • This system utilizes a central unit of lysine residues to which multiple copies of the same peptide of the invention are synthesized.
  • Each MAP contains multiple copies of one or more of the peptides of the invention.
  • One embodiment of a MAP contains at least three, but preferably four or more, peptides.
  • One skilled in the art can readily prepare any number of multimeric compounds according to the peptides identified in the above formula. All such multimeric arrangements and constructs are intended to be part of this invention. Other combinations in the form of multimers can be made on the surface of particles presenting the peptides or peptide derivatives on their surface.
  • the particle can then act as a carrier of a peptide or peptide derivative and can simultaneously act as a detectable marker.
  • multimers can be obtained by N-terminal biotinylation of the N-terminal end of the peptide or peptide derivative chains and subsequent complex formation with streptavidin. Since streptavidin can bind four biotin molecules or conjugates with high affinity, very stable tetrameric peptide complexes are obtained with this method.
  • Multimers can be prepared from identical or different peptides or peptide derivatives of the invention.
  • the multimers according to the invention preferably contain two or more peptide or peptide derivatives in which each component contributes a certain amount to the biocidal activity (target recognition, antimicrobial activity, purification).
  • Another object of this invention is the use of the peptides or peptide derivatives herein in medicine or pharmacy, eg for therapy with an antibiotic or in a composition having antimicrobial (especially bacteriocidal) activity.
  • the peptide is used medically as an antibiotic against Gram-positive bacteria.
  • the invention also relates to the peptides according to the invention for use in medicine, as an antibiotic, in a disinfectant or cleaning agent, as a preservative or in a packaging material.
  • the inventively modified peptide is particularly suitable for the treatment of microbial, bacterial or fungal infections.
  • the invention also relates to the use of the peptides according to the invention for the production of a medicament, in particular an antibiotic, in particular for the treatment of microbial infections, e.g. by bacteria, viruses and fungi.
  • the invention also relates to the use of the peptides according to the invention in pharmaceutical research or in a screening method, preferably in a screening method for the identification of substances which have an antimicrobial, bactericidal or antifungal activity.
  • a suitable such screening method for identifying a substance likely to have antimicrobial, bactericidal or antifungal activity comprises: (i) performing a competitive assay with:
  • a further subject of this invention are pharmaceutical compositions containing one or more peptides of the invention or their multimeric constructs independent of the presence of other pharmaceutically active compounds.
  • Also part of this invention is the use of the peptides of the invention as a pharmaceutical and / or for the preparation of an active ingredient which can be used as an antibiotic.
  • the peptides according to the invention can also be used individually in pharmaceutical products. Alternatively, one or more modified peptides as described above may be fused or conjugated to another compound to enhance pharmacokinetics or bioavailability without eliciting an immune response. Any number of individual peptides or multimeric constructs can be mixed together to produce a single composition.
  • a pharmaceutical composition according to the invention contains a therapeutically effective amount of one or more peptides according to the invention or their multimeric constructs. Once assembled, the pharmaceutical composition of the present invention can be administered directly to the subject to treat microbial (especially bacterial) infections. For this purpose, a therapeutically effective amount of a composition according to the invention is administered to the subject to be treated.
  • compositions of the invention are designed to treat infections of a bacterial or fungal infected mammal, including man.
  • At least one or alternatively also several peptides according to the invention or their multimeric constructs can be mixed to give an antimicrobially (in particular antibacterial or fungicidal) active composition with a pharmacologically acceptable carrier or other components.
  • the selected peptide is preferably produced synthetically or recombinantly as described above.
  • the direct administration of the pharmaceutical composition according to the invention is carried out locally or systemically, preferably orally, parenterally, intraperitoneally, intravenously, intramuscularly, pulmonarily or interstitially into the tissue.
  • the pharmaceutical composition of the invention may further contain suitable and pharmaceutically acceptable carriers, diluents, buffers or solvents and may take the form of a capsule, tablet, troche, dragee, pill, drops, suppositories, powder, spray, vaccine, ointment, paste, cream, inhalant, Plaster, aerosol or the like.
  • suitable and pharmaceutically acceptable carriers such as dispersion or suspension aids, surface-active agents, isotonic agents, thickeners or emulsifiers, preservatives, encapsulating agents, solid binders or lubricants can be used, whichever is best for the particular dosage is suitable and at the same time compatible with the peptide, peptide derivative or conjugate.
  • the pharmaceutical composition according to the invention therefore preferably contains a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier also includes a carrier for administering the therapeutic composition, such as antibodies or polypeptides, genes, or other therapeutic agents.
  • the term refers to any pharmaceutical carrier that does not itself elicit the production of antibodies which Suitable "pharmaceutically acceptable carriers” may include large, slowly degradable macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, etc.
  • Such carriers are well known to those skilled in the art.
  • Salts of the inventive peptides are prepared by known methods, which typically means that the peptides of the invention, their peptide conjugates or conjugates are mixed with a pharmaceutically acceptable acid to form an acid salt or with a pharmaceutically acceptable base to a basic salt.
  • a pharmaceutically acceptable acid to form an acid salt or with a pharmaceutically acceptable base to a basic salt.
  • an acid or a base is pharmaceutically acceptable can be readily determined by one skilled in the art, having regard to the application and formulation. For example, not all acids and bases that are acceptable for ex vivo applications are also transferable to therapeutic formulations.
  • pharmaceutically acceptable acids may be both organic and inorganic, eg formic, acetic, propionic, lactic, glycolic, oxalic, pyruvic, succinic, maleic, malonic, cinnamic, sulfuric, hydrochloric, hydrobromic, nitric, perchloric, Phosphoric acid and thiocyanic acid, which form ammonium salts with the free amino groups of peptides and functionally equivalent compounds.
  • organic and inorganic eg formic, acetic, propionic, lactic, glycolic, oxalic, pyruvic, succinic, maleic, malonic, cinnamic, sulfuric, hydrochloric, hydrobromic, nitric, perchloric, Phosphoric acid and thiocyanic acid, which form ammonium salts with the free amino groups of peptides and functionally equivalent compounds.
  • Pharmaceutically acceptable bases which form carboxylates with free carboxylic acid groups of the peptides and functionally equivalent compounds include ethylamine, methylamine, dimethylamine, triethylamine, isopropylamine, diisopropylamine, and other mono-, di-, and trialkylamines, as well as arylamines. Also included are pharmaceutically acceptable solvents.
  • salts of mineral acids such as hydrochlorides, hydrobromides, phosphates, sulfates and the like; but also salts of organic acids, such as acetates, propionates, malonates, benzoates and the like.
  • compositions of the invention further include liquids, for example, water, salt water, glycerol, and ethanol.
  • pharmaceutical compositions of the invention may contain adjuvants, such as humectants or emulsifiers, pH buffering substances, and similar compounds.
  • the pharmaceutical compositions of the invention are prepared either in liquid form or as a suspension for injection, solid forms for dissolving or suspending in carrier fluids prior to injection are also possible.
  • liposomes are encompassed by the definition of a "pharmaceutically acceptable carrier”.
  • peptides of the invention or their peptide conjugates can be produced and administered to a subject in need thereof.
  • the peptide or peptide conjugate may be administered to a subject in any suitable form, preferably as a pharmaceutical composition adapted to the dosage form and present in a dosage appropriate for the desired treatment.
  • the pharmaceutical compositions of this invention may contain other active compounds, for example, conventional antibiotics (eg, vancomycin, streptomycin, tetracycline, penicillin) or other antimicrobial active compounds such as fungicides, eg, intraconazole or myconazole.
  • Other compounds that alleviate symptoms associated with the infection such as fever (salicylic acid) or rash, may also be added.
  • peptides or peptide derivatives according to the invention in disinfectants or cleaning agents (for example a bacteriocidal composition) which can be used for disinfecting or cleaning surfaces or objects, in particular Avoidance or removal of biofins.
  • a bacteriocidal composition for example a bacteriocidal composition
  • Another field of application is packaging where peptides according to the invention can be bound to or incorporated into packaging material, or as preservatives for other materials that can be readily degraded by microorganisms.
  • the peptides or peptide derivatives according to the invention are particularly suitable for the packaging of foodstuffs, since they have no toxic effect on contact or on ingestion.
  • Another component of this invention is a method of treating mammals infected with microbes (especially bacteria or fungi), including administering an effective, therapeutically effective amount of the pharmaceutically active composition of the invention.
  • the term "therapeutically effective amount” refers to the amount of a therapeutic, ie, a peptide, peptide derivative or conjugate of the invention, which can reduce or completely prevent the multiplication and colonization of the bacteria or achieve measurable therapeutic or prophylactic success.
  • the effect can be determined, for example, for biopsies in culture, by testing bacterial activity, or by another suitable method for assessing the extent and degree of bacterial infection.
  • the exact effective amount for a subject depends on its size and condition, the species and
  • the pharmaceutical compositions of the invention may be used to treat bacterial infections and / or biological or physical conditions reduce or prevent (e.g., fever).
  • Methods for determining the starting dose by a physician are state of the art. The specified doses must be safe and successful.
  • the amount of a peptide of the present invention necessary for an antibacterially effective dose may take into account the pathogen causing the infection, the severity of the infection, as well as the age, weight, sex, general physical condition, etc. of the patient Patients are determined.
  • the necessary amount of the peptide according to the invention to be effectively effective antibacterial and antimycotic without significant side effects depends on the pharmaceutical formulation used and the eventual presence of other ingredients such as. Antibiotics, antimycotics, etc. from.
  • an effective dose may be between 0.01 nmol / kg and 50 nmol / kg, preferably between 0.2 nmol / kg and 10 nmol / kg of the peptide, peptide derivative or conjugate in the treated individual.
  • Initial doses of the peptides, peptidomimetics, multimers, peptide conjugates or peptidomimetics conjugates of the invention may optionally be monitored by repeated administration.
  • the frequency of dosages depends on the factors identified above and is preferably between one and six doses per day over a treatment period of about three days to a maximum of one week.
  • the compounds are administered pulmonarily in a certain amount, e.g. through an inhaler, nebulizer, aerosol spray or a dry powder inhaler.
  • Suitable formulations can be prepared by known methods and techniques. Transdermal or rectal delivery may be appropriate in some cases, as well as administration to the eye.
  • the dosage form may contain any substance or mixture that enhances bioavailability. This can be achieved, for example, by reducing the degradation, e.g. by an enzyme inhibitor or an antioxidant. It is better if the bioavailability of the compound is achieved by an increase in the permeability of the absorption barrier, usually the mucous membrane. Substances that facilitate penetration can act in different ways; some increase the fluidity of the mucosa, while others expand the spaces between the mucosal cells. Still others reduce the viscosity of the mucus on the mucous membrane.
  • the preferred accelerators include amphiphilic substances such as cholic acid derivatives, phospholipids, ethanol, fatty acids, oleic acid, fatty acid derivatives, EDTA, carbomers, polycarbopbil and chitosan.
  • Indications for which the modified peptides, their conjugates or multimers of the invention can be used are bacterial infections with both Gram-positive and Gram-negative bacteria, for example Escherichia coli, Enterobacter cloacae, Erwinia amvlovora, Klebsiella pneumoniae, Morganella morganii, Pseudomonas aeruginosa Salmonella typhimurium, Salmonella typhi, Shigella dysenteriae, Yersinia enterocolitica, Acinetobacter calcoaceticus, Acinetobacter banmannii, Agrobacterium tumefaciens, Francisella tularensis, Legionella pneumophila, Pseudomonas syringae, Pseudomonas aeruginosa, Rhizobium meliloti, Haemophilus influenzae and Staphylococcus aureus.
  • Escherichia coli Enterobacter cloaca
  • Fig. 1 shows the results of the substitution analysis of Apidaecin Ib (GNNRPVYIPQPRPPHPRL - SEQ ID NO: 2) with the respective amino acids given in one-letter code. Values below 1 indicate the improvement in microbial activity. Values above 1 for deterioration.
  • the left column shows the native sequence. The upper line shows the substituent. For example, the value of 0.55 just below the C is a measure of the activity of the native apidaecin sequence substituted at position 1 with cysteine.
  • Fig. 2 shows a particular embodiment of the peptides described herein.
  • Each peptide labeled with X is more effective than the native apidaecin and particularly advantageous against Gram-positive bacteria.
  • Fig. 3 shows a permeability assay.
  • Api88 corresponds to the peptide with SEQ ID NO. 92;
  • Apil 37 corresponds to the peptide with SEQ ID NO. 93;
  • Api341 corresponds to the peptide with SEQ ID NO. 89th
  • substitution analysis was carried out with this peptide (according to SEQ ID No. 2).
  • the substitution library was synthesized and tested for antibacterial activity against Pseudomonas aeruginosa using the bioluminescence assay.
  • the SPOT synthesis of the peptide libraries was carried out on a Whatman 50 filter paper cutout (Sigma-Aldrich, Germany) of the size 19 ⁇ 29 cm using the Fmoc method and a SPOT synthesizer (Intavis AG, Germany) (corresponding to Reineke U et al., 2001).
  • the luminescence screening method used is based on the publication by Hilpert and Hancock (Hilpert, 2007.
  • the peptides synthesized on the membrane were cleaved off and the peptide spots were punched out of the peptide membrane using a hole punch and placed in a 96-well microtiter plate (Corning, USA transferred) and Per well least 200 ⁇ ⁇ . water was added.
  • each peptide in the array was to transmit exactly one well of a microtiter plate
  • the sealed master plates were stored at -20 ° C. The master plates were designed so that each row contains 10 peptides and two controls (positive and negative).
  • the actual screening took place.
  • an overnight culture 37 ° C., 225 rpm, 18 h
  • the overnight culture was diluted 100-fold and grown to the optical density of 0.35 at 600 nm [OD600] (approximately 2 hours - logarithmic phase culture - LogK).
  • the incubation suspension (4 vol.% LogK in 100 mM Tris-HCl buffer (pH 7.3) with 40 mM sterile-filtered glucose) was then distributed on luminescence-suitable 96-well plates (VWR, Germany) and with a concentration series of the peptide library for 4 h long incubated at 37 ° C. After incubation, the luminescence was measured using the luminometer (Thermo, Finland).
  • the peptide sequences were selected which showed the highest activity in the assay. These peptides were conventionally synthesized on a polymeric support and assayed for their antibacterial activity against P. aeruginosa, E. coli, and S. aureus by the MIC assay.
  • Example 2 Determination of minimum inhibitory concentrations and growth kinetics
  • the minimum inhibitory concentrations (MIC) of the peptides were determined in a double determination of triplicates with a positive (gentamycin) and a negative control (0.9% NaCl solution) according to a modified protocol from Wiegand et al. (Wiegand, 2008).
  • the peptides were dissolved in water and diluted in duplicate with 1/8 MH (eight times diluted Mueller-Hinton medium - 2.6 g / L, Merck) in sterile 96-well plates (Greiner Bio-One GmbH) 128 ⁇ g / mL diluted in twelve dilution steps to 62.5 ng / mL. Overnight cultures were set at 1/8 MHB to approximately 1.5 x 10 7 colony forming units per mL. Of these, 50 peptide solutions per well each with 50 of the Bacteria solution mixed to reach an initial concentration of 4 x 10 5 bacteria per well. After incubation for 20 hours at 37 ° C, the absorbance at 595 nm (microplate reader, Wallac Victor3, Perkin Elmer) was determined. The minimal inhibitory concentration was identified as the lowest peptide concentration at which no bacterial growth was detected.
  • the antibacterial activity of the peptides according to the invention was analyzed against the following bacterial strains: Pseudomonas aeruginosa PAOL (wt strain), Pseudomonas aeruginosa DSM 9644, Staphylococcus aureus DSM 1 104 / ATCC 25923, Staphylococcus aureus ATCC 6247, Escherichia coli UB1005 (F, nalA37 , metBl) and Escherichia coli ATCC25922.
  • Table 4 below shows the results of the experiment:
  • the antibacterial activity of the peptides according to the invention was analyzed against the following pathogenic bacterial strains of the Gram-positive bacterium S. aureus and P. aeruginosa:
  • PBS phosphate buffered saline (English: Phospho-buffered saline)

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  • Chemical & Material Sciences (AREA)
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  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Insects & Arthropods (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des peptides antibiotiques modifiés, s'utilisant en particulier en médecine. L'invention concerne des compositions et des méthodes utilisées pour la destruction de micro-organismes comme des bactéries, des virus ou des champignons ainsi que des méthodes pour traiter des infections microbiennes. L'invention vise à mettre au point de nouveaux peptides antibiotiques, en particulier à effet antibiotique amélioré et à spectre d'action élargi vis-à-vis de souches de bactéries, en particulier de bactéries gram positif, comme le staphylocoque doré. Selon l'invention, dans un premier aspect, le problème posé est résolu par un peptide selon la revendication 1.
EP12774981.0A 2011-09-22 2012-09-21 Dérivés d'apidaecine utilisés comme peptides antibiotiques Not-in-force EP2750689B1 (fr)

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WO2013041663A2 (fr) 2011-09-22 2013-03-28 Amp-Therapeutics Gmbh Dérivés d'apidaecine utilisés comme peptides antibiotiques
AU2016353073A1 (en) 2015-11-10 2018-06-07 Visterra, Inc. Antibody molecule-drug conjugates that specifically binds to lipopolysaccharide and uses thereof
KR101847051B1 (ko) * 2016-04-26 2018-04-09 한국외국어대학교 연구산학협력단 병원균에 대한 항생활성을 갖는 펩타이드 및 이를 포함하는 항생 펩타이드 조성물
US11890319B2 (en) * 2017-01-18 2024-02-06 Visterra, Inc. Antibody molecule-drug conjugates and uses thereof
US11969476B2 (en) 2020-04-03 2024-04-30 Visterra, Inc. Antibody molecule-drug conjugates and uses thereof
CN114539359B (zh) * 2022-03-22 2022-11-04 中国农业科学院蜜蜂研究所 一种防治蜜蜂幼虫白垩病的抗菌肽、制备方法及其应用

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EP0299828B1 (fr) 1987-07-01 1994-06-15 Plant Genetic Systems N.V. Peptides bactériocidiques et/ou bactériostatiques, procédé pour leur isolation, production et utilisation
US5466671A (en) * 1994-03-02 1995-11-14 Sloan-Kettering Institute For Cancer Research Apidaecin-type peptide antibiotics with improved activities and/or different antibacterial spectrum
DE102007036128A1 (de) * 2007-07-23 2009-02-12 Universität Leipzig Antibiotische Peptide
DE102009007381A1 (de) * 2009-01-29 2010-08-05 Amp-Therapeutics Gmbh & Co. Kg Antibiotische Peptide
DE102011118029A1 (de) * 2011-06-20 2012-12-20 Universität Leipzig Modifizierte antibiotische Peptide mit variabler systemischer Freisetzung
WO2013041663A2 (fr) 2011-09-22 2013-03-28 Amp-Therapeutics Gmbh Dérivés d'apidaecine utilisés comme peptides antibiotiques

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WO2013041663A2 (fr) 2013-03-28
EP2750689B1 (fr) 2019-08-21
WO2013041663A3 (fr) 2013-07-04
US20150344524A1 (en) 2015-12-03
US20170107256A1 (en) 2017-04-20
US10435437B2 (en) 2019-10-08

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